WO2012144799A2 - 활성 폴리펩타이드의 결합 친화력 및 결합 특이성을 증강시키는 단백질 골격 모듈 - Google Patents

활성 폴리펩타이드의 결합 친화력 및 결합 특이성을 증강시키는 단백질 골격 모듈 Download PDF

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WO2012144799A2
WO2012144799A2 PCT/KR2012/002958 KR2012002958W WO2012144799A2 WO 2012144799 A2 WO2012144799 A2 WO 2012144799A2 KR 2012002958 W KR2012002958 W KR 2012002958W WO 2012144799 A2 WO2012144799 A2 WO 2012144799A2
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protein
polypeptide
present
embedded
active
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PCT/KR2012/002958
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English (en)
French (fr)
Korean (ko)
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WO2012144799A3 (ko
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차기원
소인섭
김소연
이병헌
김인산
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경북대학교 산학협력단
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Priority to JP2014506324A priority Critical patent/JP6050319B2/ja
Priority to CN201280029758.7A priority patent/CN103703022A/zh
Priority to EP12773577.7A priority patent/EP2700649B1/en
Publication of WO2012144799A2 publication Critical patent/WO2012144799A2/ko
Publication of WO2012144799A3 publication Critical patent/WO2012144799A3/ko
Priority to US14/057,654 priority patent/US8975369B2/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5406—IL-4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Protein backbone modules that enhance binding affinity and binding specificity of active polypeptides
  • the present invention relates to a novel protein backbone module that enhances the binding affinity or binding specificity of an active polypeptide, consisting of 1 to 19th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 Polypeptides; Polypeptides including active polypeptides; And a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 1 and a 29 th to 86 th amino acid sequence.
  • a domain is a structurally functional independent unit. The same domain may be distributed more than one in several proteins, and one protein may consist of several domains. Specific information on the domains includes Prosite (Hulo N et al., Nucleic Acids Res, 36: D245-249, 2008; Website: http://expasy.org/prosite/), SMART (Letunic I et al., Nucleic Acids Res, 34: D257-D260, 2006; Website: http://smart.embl—heidelberg.de/).
  • Biomolecular interactions eg protein-protein, protein-nucleic acid, etc.
  • biomolecular interactions eg protein-protein, protein-nucleic acid, etc.
  • proteins whole or fragments
  • the problems of the antibody are low expression amount, low solubility, animal cell expression cell line, high purification cost, low stability in a reducing intracellular environment, and the like.
  • the present inventors replace the existing peptide preparations and antibody preparations by selecting a part of human-derived protein, optimally modifying them, and inserting an active peptide, thereby enhancing the binding affinity or binding specificity of the active polypeptide.
  • the present invention has been found by showing excellent properties that can be achieved.
  • Another object of the present invention is a) a polypeptide consisting of the first to 19th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1;
  • a protein-skeletal module embedded with an active polypeptide comprising a polypeptide in which a polypeptide consisting of the 29th to 86th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1 is sequentially linked. Is to provide.
  • Another object of the present invention is to provide a polynucleotide encoding the protein backbones in which the active polypeptide is embedded.
  • Another object of the present invention is to provide an expression vector comprising the polynucleotide.
  • Another object of the present invention is to provide a host cell transformed with the expression vector.
  • Another object of the present invention is to provide a method for preparing protein backbones embedded with an active polypeptide comprising culturing the host cell.
  • Another object of the present invention is to provide a use of the protein backbones embedded with the active polypeptide for the preparation of a diagnostic agent.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the protein scaffolds in which the active polypeptide is embedded as an active ingredient.
  • Another object of the present invention is to provide a diagnostic kit comprising the protein skeleton of the active polypeptide embedded therein as an active ingredient.
  • Another object of the invention is a) a polypeptide consisting of the first to 19th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1;
  • polypeptide comprising an active polypeptide comprising a polypeptide consisting of polypeptides consisting of the 29th to 86th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1 in sequence; will be.
  • Another object of the present invention is to provide a polynucleotide encoding the protein backbones in which the active polypeptide is embedded.
  • Another object of the present invention is to provide a composition for diagnosing cancer or atherosclerosis comprising the protein skeleton of the active polypeptide embedded therein as an active ingredient.
  • Another object of the present invention is to provide a method for diagnosing cancer or arteriosclerosis, comprising administering to the subject in need thereof an effective amount of the protein backbone embedded with the active polypeptide.
  • Another object of the present invention is to provide a use of the protein backbone module containing the active polypeptide for the preparation of a diagnostic agent for cancer or atherosclerosis.
  • Another object of the present invention is to provide a kit for diagnosing cancer or atherosclerosis comprising an active polypeptide-containing protein backbone as an active ingredient.
  • the present invention provides a polypeptide comprising: a) a polypeptide consisting of the 1st to 29th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1; b) a polypeptide comprising an active polypeptide and
  • c) Provides protein backbones embedded with an active polypeptide, characterized in that the polypeptide consisting of the amino acid sequence of the 29th to 86th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 comprises a polypeptide connected in sequence.
  • the present invention provides a polynucleotide encoding the protein backbone parent embedded in the active polypeptide.
  • the present invention provides an expression vector comprising the polynucleotide.
  • the present invention provides a host cell transformed with the expression vector.
  • the present invention provides a method for preparing protein backbones embedded with an active polypeptide comprising culturing the host cell.
  • the present invention provides a diagnostic composition comprising the protein skeleton of the active polypeptide is embedded as an active ingredient.
  • the present invention provides a diagnostic method comprising the step of administering an effective amount of the protein backbone parent embedded in the active polypeptide to an individual in need thereof.
  • the present invention provides the use of the protein backbone, embedded in the active polypeptide for the preparation of a diagnostic agent.
  • the present invention provides a pharmaceutical composition comprising a protein backbone module containing the active polypeptide as an active ingredient.
  • the present invention provides a diagnostic kit comprising the protein skeleton of the active polypeptide is embedded as an active ingredient.
  • the present invention provides a polypeptide comprising: a) a polypeptide consisting of the 1 st to 19 th amino acid sequences of an amino acid sequence represented by SEQ ID NO: 1; b) a polypeptide comprising the amino acid sequence of activator protein 1 (AP-l) represented by SEQ ID NO: 3 and
  • a polypeptide comprising an active polypeptide comprising a polypeptide consisting of polypeptides consisting of the 29th to 86th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1 is sequentially linked.
  • the present invention provides a polynucleotide encoding the protein backbone parent embedded in the active polypeptide.
  • the present invention provides a composition for diagnosing cancer or atherosclerosis comprising the active polypeptide-containing protein backbone moduleol active ingredient.
  • the present invention provides a method for diagnosing cancer or arteriosclerosis, comprising administering to a subject in need thereof an effective amount of the protein backbone module containing the active polypeptide. .
  • the present invention provides a use of the protein backbone of the active polypeptide embedded in the manufacture of a diagnostic agent for cancer or atherosclerosis.
  • the present invention provides a kit for diagnosing cancer or arteriosclerosis comprising an active polypeptide-containing protein skeletal hair as an active ingredient.
  • the present invention relates to a polypeptide comprising: a) a polypeptide consisting of the 1st to 19th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1; an active polypeptide comprising a polypeptide comprising: b) a polypeptide comprising an active polypeptide and c) a polypeptide consisting of the 29th to 86th amino acid sequences of the amino acid sequence represented by SEQ.
  • the protein backbone module of the present invention is characterized by enhancing the binding affinity or binding specificity of the active polypeptide embedded therein. .
  • the active polypeptide refers to a polypeptide that performs various functions such as signaling, activation, deactivation, and phosphorylation of the target protein through contact with the target protein.
  • the protein backbone hairs of the present invention increase the activity of an active polypeptide in a manner that increases the binding affinity or binding specificity of the embedded active polypeptide. Binding affinity refers to the strength of a property to bind to in vivo molecules.
  • Binding specificity refers to a property in which a specific biomolecule is specifically bound to a molecule to be bound.
  • the protein backbone hairs of the present invention refer to a protein construct capable of embedding an active polypeptide without affecting the immune system in vivo.
  • Protein skeletal hairs of the present invention have a low molecular weight, have excellent activity in vivo, do not affect the activity of the embedded active polypeptide, do not affect the immune system in vivo, and are present in abundance in vivo. There should be no differences in individual effects from the protein and no occurrence of individual specific effects.
  • a protein backbone module that incorporates an active polypeptide to improve the stability and properties of the active polypeptide was first developed by the present inventors and was named DMID (Designed Modular Immunodi agnostics).
  • the active polypeptide of the present invention may have pharmacological activity, and protein backbones in which the active polypeptide is embedded may exhibit inherent pharmacological activity due to the action of the active polypeptide.
  • protein backbones in which the active polypeptide is embedded may have the pharmacological activity of the active polypeptide, in particular, the pharmacological activity of the enhanced effect.
  • the protein backbone hairs of the present invention are preferably designed based on a Stabilin-2 derived polypeptide fragment, more preferably active on the part of the amino acid sequence represented by SEQ ID NO: 1 that does not affect the backbone.
  • the polypeptide may be a protein backbone with substituted or inserted.
  • Stabilin-2 is a transmembrane receptor that is involved in lymphocyte induction, cell adhesion, receptor scavenging, and angiogenesis. This protein has a variety of domains including 7 fasciclin, 16 epidermal growth factor (EGF) -like, 2 laminin-type EGF-1 ike domains, a C to type lectin ⁇ like hya 1 ur onan-b i nd i ng Link module Have.
  • EGF epidermal growth factor
  • the present invention provides a polar nucleotide encoding the protein backbone.
  • the polynucleotide of the present invention is characterized by encoding protein backbone moieties that enhance the binding affinity or binding specificity of the active polypeptide.
  • a polynucleotide of the present invention may be a substance encoding a protein backbone having an amino acid sequence represented by SEQ ID NO: 1 or having an amino acid sequence having at least 70% homology with the amino acid sequence.
  • the polynucleotide may be DNA or RNA, preferably DNA represented by SEQ ID NO: 2.
  • the present invention is characterized by providing an expression vector into which the polynucleotide of the present invention is inserted.
  • the expression vector is an expression vector prepared by those skilled in the art to express the protein backbone of the present invention by inserting the polynucleotide of the present invention into the vector according to any method known in the art using an appropriate transcription / translation control sequence. Say.
  • expression vector refers to a plasmid, virus or other media known in the art in which a polynucleotide sequence encoding a protein of the present invention can be inserted or introduced.
  • the polynucleotide sequence according to the present invention may be operably linked to an expression control sequence, wherein the operably linked gene sequence and the expression control sequence comprise one expression vector containing a selection marker and a replication origin. It may be included within.
  • “Operably linked” can be genes and expression control sequences linked in such a way that enables expression of the gene when the appropriate molecule is bound to the expression control sequence.
  • an "expression control sequence” refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular host cell.
  • Such regulatory sequences include promoters for performing transcription, any operator sequence for controlling transcription, sequences encoding suitable m NA ribosomal binding sites, and sequences that control the termination of transcription and translation.
  • Expression vectors capable of inserting the polynucleotide encoding the protein backbone of the present invention include E. coli-derived plasmids (pBR322, pBR325, pUC118, pUC119, pET30a, pET30c and pGEX-GST), derived from Bacillus subtilis Plasmids (pUBllO and ⁇ 5), yeast derived plasmids ( ⁇ 13, ⁇ 24 and YCp50) and Ti plasmids, and animal viruses such as retroviruses, adenoviruses or vaccinia viruses.
  • E. coli-derived plasmids pBR322, pBR325, pUC118, pUC119, pET30a, pET30c and pGEX-GST
  • Bacillus subtilis Plasmids pUBllO and ⁇ 5
  • yeast derived plasmids ⁇ 13, ⁇ 24 and YCp50
  • Scion viruses and plant viruses such as sucrose and baclovirus can be used, and binary vectors such as the pPZP, pGA and pCAMBIA families can be used.
  • Those skilled in the art can select a vector suitable for introducing the polynucleotide sequence of the present invention, and any vector may be used as long as the vector can dop the polynucleotide sequence of the present invention into a host cell.
  • a vector designed to facilitate protein expression induction and isolation of the expressed protein may be used, and more preferably, a pET32a vector, which is a recombinant vector comprising the polynucleotide of the present invention, may be used.
  • the present invention also provides a host cell transformed with the recombinant expression vector of the present invention.
  • the transformed host cell may be a microorganism transformed by a person skilled in the art using a recombinant expression vector of the present invention according to a method known in the art, and the microorganism is preferably, but not limited to, E. coli ( E. COli) and most preferably E. coli BL21 or E. coli MC 1061.
  • the transformation of the recombinant vector by introducing the vector into the host cell according to the present invention may include, but is not limited to, calcium chloride (CaCl 2 ), a heat shock method, and a particle gun method. bombardment, silicon carbide whiskers, sonication, e'lectroporation, and precipitation by polyethylene glycol (PEG) can be used.
  • CaCl 2 calcium chloride
  • PEG polyethylene glycol
  • host-expressing vector systems can be utilized to express the protein backbone models of the invention.
  • Such host-expression systems provide a vehicle capable of generating and subsequently purifying the desired coding sequence, and upon transformation or transfection with the appropriate nucleotide coding sequence, the protein backbone moieties of the invention in situ. It means a cell which can be expressed.
  • Such systems include, but are not limited to, bacteria transformed with microorganisms such as recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the protein backbone mode coding sequences of the invention (e.g., E. coli and B.
  • Yeast infected with recombinant yeast expression vectors containing protein backbone mode coding sequences eg, Saccharomyces, Pichia
  • Nasal cell systems infected with recombinant virus expression vectors (eg, baclovirus) containing protein backbone mode coding sequences eg, baclovirus
  • a recombinant plasmid expression vector eg Ti plasmid
  • Transgenic or recombinant expression vectors eg, cauliflower mosaic virus
  • CaMV Tobacco mosaic virus, TMV
  • TMV tobacco mosaic virus
  • a promoter derived from the genome of a mammalian cell eg a metallothionein promoter
  • a promoter derived from a mammalian virus eg adenovirus late promoter; a vaccinia virus 7.5K promoter
  • Mammalian cell systems carrying recombinant expression constructs eg, COS, CHO, BHK, 293, NS0 and 3T3 cells).
  • the protein backbone module of the present invention is characterized by enhancing the binding affinity or binding specificity of the built-in active polypeptide, and the incorporation of such active polypeptide encodes an active polypeptide to be embedded in the protein backbone module. It may be a method of inserting and expressing a polynucleotide encoding an active polypeptide at a specific site of a nucleotide.
  • the present invention also provides an expression vector comprising a polynucleotide encoding the protein backbone module in which the active polypeptide is embedded and a host cell transformed with the expression vector.
  • the expression vector and the host cell are as described above.
  • the host cell may be preferably E. COl i. .
  • the present invention provides a method for preparing protein skeletal hairs embedded with an active polypeptide comprising culturing the host cell.
  • protein backbone hairs embedded with an active polypeptide having enhanced binding affinity or binding specificity can be efficiently produced.
  • the protein backbone module embedded with the active polypeptide prepared by the method of the present invention may be prepared by any method known in the art for protein purification, for example, chromatography (e.g., metal-chelate chromatography). Chromatography, ion exchange affinity and size column chromatography, etc.), centrifugation, column solubility or any other standard method for protein purification.
  • Protein backbones embedded with an active polypeptide having enhanced binding affinity or binding specificity of the present invention are more resistant to target substances than the active polypeptide alone. ⁇ High affinity or binding specificity.
  • the protein backbone of the present invention may be further attached to the labeling material used in the known detection method.
  • protein backbone of the present invention can be further attached to a substance or the like effective to the target of the binding of the active polypeptide.
  • the protein backbones of the present invention include polyethylene glycol (PEG), human serum albumin (HSA), Fc regions of antibodies, IgG molecules, cytotoxic drugs, radioisotopes, contrast agents, His-tags, biotin, Flag-tags, nucleic acids, cytokines and the like can be further attached.
  • the present invention provides a diagnostic composition comprising the protein backbones in which the active polypeptide is embedded as an active ingredient.
  • the present invention provides a diagnostic method comprising the step of administering an effective amount of the protein backbone embedded with the active polypeptide to a subject in need thereof.
  • the present invention also provides the use of the protein backbones embedded with the active polypeptide of claim 1 for the preparation of a diagnostic agent.
  • the term 'effective amount' refers to an amount that exhibits an effect of diagnosing or providing information necessary for diagnosing a disease in a subject to which the composition or formulation of the present invention is administered.
  • the subject may be a patient in need of diagnosis.
  • composition of the present invention is a protein backbone module embedded with the active polypeptide
  • It may have a composition comprising 0.001 to 99.999% by weight and the balance of the carrier.
  • the diagnostic composition in the present invention is characterized in that it comprises a protein skeleton of the active polypeptide is embedded in the present invention as an active ingredient.
  • Diagnosis in the present invention can determine the presence, distribution, amount, etc. of the target substance of the active polypeptide, and means to determine the presence and progress of the disease associated with it.
  • the IL-4 receptor is overexpressed in various human cancers (colon cancer, lung cancer, uterine cancer and breast cancer). Since the protein skeleton of AP-1 of the present invention has binding ability to IL-4 receptor, it can be used for diagnosis and treatment of the cancer.
  • Methods for determining the presence and amount and / or pattern of proteins include Western blets, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmuno unodi f fusion. ), Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc. There is, but is not limited to this.
  • measurement can be made by detecting the magnitude and pattern of a signal of a detection label usually associated with a secondary antibody.
  • Such detection labels may include, but are not limited to, enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, radioisotopes, and the like.
  • enzymes include ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Esterases, glucose oxidases, nucleokinase and GDPase, RNase, glucose oxidases, luciferases, phosphofructokinases, phosphoenolpyruvate carboxylase, aspartate aminotransferases, phosphphenolpyruvates Decarboxylase, ⁇ -latamase, and the like, but are not limited thereto.
  • fluorescent materials include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, 0-phthalaldehyde, and fluorescamine. And the like, but are not limited thereto.
  • available ligands include, but are not limited to, biotin derivatives.
  • available luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like.
  • the microparticles are used as the detection label, the microparticles available include, but are not limited to, colloidal gold and colored latex.
  • the available redox molecules include Ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4W (CN) 8 , [0s (bpy) 3 ] 2+ , [RU (bpy ) 3 ] 2+ , [M0 (CN) 8 ] 4 —and the like, but are not limited to this.
  • the diagnostic composition of the present invention can determine the presence, distribution, amount, etc. of the target material of the active polypeptide, and provides information for determining the presence, progress of the disease associated with it.
  • the composition of the present invention is excellent in diagnostic sensitivity, accuracy and accuracy compared to the diagnostic composition containing an active polypeptide that is not embedded in the protein backbone module of the present invention. Therefore, the present invention provides a diagnostic kit comprising the protein backbones in which the active polypeptide is embedded as an active ingredient.
  • the diagnostic kit may further include a support, a suitable complete solution, a secondary antibody labeled with a chromophore or a fluorescent substance, a chromogenic substrate solution, and the like.
  • the support may be a nitro sal rhodium membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, or a glass slide glass, wherein the color developing enzyme is a peroxidase. (peroxidase), or alkaline phosphatase (alkal ine phosphatase) can be rounded, the fluorescent material may be FITC, or RITC, etc.
  • the chromogenic substrate solution is ABTS (2,2'-Az K) -bis (3 -ethylbenzenzothiazoline-6-sulfonic acid), o-Phenylenediamine (OPD), or TMB Tetramethyl Benzidine).
  • the present invention also provides a pharmaceutical composition comprising as an active ingredient the protein backbones in which the active polypeptide is embedded. ⁇
  • the pharmaceutical composition of the present invention can deliver effective therapeutic substances, cytotoxic drugs, etc. to the binding target of the active polypeptide.
  • the pharmaceutical composition depends on the type of active polypeptide and its target disease Can be.
  • IL-4 receptor is overexpressed in various human cancers (colon cancer, lung cancer, uterine cancer and breast cancer). As it binds to, it is possible to deliver therapeutic substances or cytotoxic drugs that target it.
  • the present invention provides a polypeptide comprising: a) a polypeptide consisting of the 1 st to 19 th amino acid sequences of an amino acid sequence represented by SEQ ID NO: 1;
  • AP-1-embedded protein backbone module comprising a polypeptide in which the polypeptide consisting of the 29th to 86th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 is sequentially connected.
  • the polynucleotide may be any one selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 6.
  • the active polypeptide refers to a polypeptide that performs various functions such as activation, deactivation, and phosphorylation of a signaling target protein through contact with the target protein.
  • AP-1 has an amino acid sequence that the cytokine IL-4 binds to the IL-4 receptor, and thus binds itself to the IL-4 receptor. Since IL-4 receptor is known to be overexpressed in various human cancers (colon cancer, lung cancer, uterine cancer and breast cancer), the development of an active polypeptide that selectively binds to IL-4 receptor can be useful for the diagnosis and treatment of cancer. have. Since IL-4 is a cytokine that increases cell adhesion, and acts to induce atherosclerosis, AP-1 peptide can be developed as a diagnostic and therapeutic agent for atherosclerosis.
  • the present invention is for cancer or arteriosclerosis diagnostic composition comprising a protein skeletal hair embedded in AP-1 as an active ingredient and cancer or arteriosclerosis diagnostic comprising a protein skeletal hair embedded in AP-1 as an active ingredient.
  • a kit comprising
  • the present invention provides a method for diagnosing cancer or atherosclerosis, comprising administering an effective amount of AP-1-embedded protein backbone to an individual in need thereof.
  • the present invention is a built-in AP-1 for the manufacture of a diagnostic agent for cancer or atherosclerosis Provides the use of protein backbone models.
  • the cancer may be any cancer as long as the cancer changes the level of a biomaterial having a structure having affinity for AP-1 by its occurrence, progression, degeneration, or extinction.
  • the cancer is colon cancer, lung cancer, uterine cancer, It may be breast cancer.
  • the pharmaceutical composition according to the present invention may contain the protein backbone module of the present invention alone or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents.
  • Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration.
  • Carriers for oral administration may include methose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may include various drug delivery materials used for oral administration to the peptide formulation.
  • carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose, glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol.
  • Other pharmaceutically acceptable carriers may be referred to those described in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, East on, PA, 1995.
  • composition of the present invention can be administered to any mammal, including humans, by any method.
  • it can be administered orally or parenterally.
  • Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration. Can be.
  • composition of the present invention may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above.
  • the composition of the present invention may be a powder, granules, tablets, pills, sugar tablets, capsules, solutions, gels, syrups, slurries, suspensions, etc.
  • oral preparations contain the active ingredients in Tablets or dragees can be obtained by blending with the brothers and then grinding it and adding suitable auxiliaries and processing into granule mixtures.
  • suitable excipients include sugar and corn starch, wheat starch, rice starch and potato starch, including lactose, dextrose, sucrose, solbi, manny, xili, erysri and malty.
  • Layered agents such as starch, cellulose, methyl cellulose, sodium carboxymethyl cellulose and hydroxypropylmethyl-salrose, and the like, including cellulose, gelatin, polyvinylpyridone, and the like. .
  • crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant.
  • the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier and a preservative.
  • parenteral administration it may be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants in the art. These formulations are described in Remington's Pharmaceutical Science, 15th Edit ion, 1975. Mack Publishing Company, East on, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a prescription generally known in all pharmaceutical chemistries.
  • the total effective amount of the protein backbone module of the composition of the present invention may be administered to a patient in a single dose, and may be administered in a fractionated treatment protocol that is administered for a long time in multiple doses. May be administered.
  • the pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the extent of the disease.
  • the preferred dose of protein skeletal wool of the composition of the present invention may be from about O.Olug to l, 000mg, most preferably O.lug to lOOmg per kg of patient body weight per day.
  • the dosage of the protein skeletal mothers is determined by the effective dosage for the patient in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet and the rate of excretion, as well as the route of administration and the frequency of treatment of the pharmaceutical composition.
  • the pharmaceutical composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is shown. Such effects of the present invention are well illustrated in the examples of the present specification.
  • Stamiin-2 is the most suitable domain by screening modular domains having high affinity, low molecular weight and immunogenicity, abundant presence in humans, and validating three-dimensional structure. EGFL domain of was selected. Three-dimensional modeling of the selected sequences and mutations on in silico established the most suitable model.
  • a polynucleotide sequence encoding selected protein backbones was prepared, introduced into an expressible expression vector, and transformed into E. coli.
  • the transformed strains were cultured to express the protein skeletal hairs of the present invention and then extracted and purified.
  • the surface plasmon resonance analysis was used to measure the binding capacity of the AP-1-embedded protein backbone as an active polypeptide of the present invention.
  • the affinity is 174 times higher than the naked AP-1 peptide.
  • the detection sensitivity change of the active polypeptide by the protein backbone of the present invention was measured.
  • AP-1 is embedded in the protein backbone module of the present invention, it was confirmed that the activity of IL-4 was well inhibited 10 times or more as compared to the naked AP-1 peptide.
  • the diagnostic sensitivity was measured by the protein backbone module incorporating the active polypeptide of the present invention.
  • the tumor targeting function was improved when the AP ⁇ 1 was embedded in the protein skeleton of the present invention compared to the naked AP-1 peptide.
  • the present invention relates to a novel protein backbone model that enhances the binding affinity or binding specificity of an active polypeptide, and more specifically, the first to 19th amino acid sequences represented by SEQ ID NO: 1.
  • This amino acid sequence Loosened polypeptide; Polypeptides including active polypeptides; And a polypeptide consisting of the 29 th to 86 th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1.
  • the protein backbone hairs of the present invention enhance the binding affinity or binding specificity of the built-in active polypeptide and thus are effective in the diagnosis and treatment of diseases.
  • Figure 1 is a schematic diagram of the domain arrangement of Stabilin-2 and three-dimensional modeling results of the protein backbone module and the protein backbone module embedded with the active peptide of the present invention.
  • Figure 2 is a graph of the surface plasma resonance experiment results (DMID AP-1: of the present invention
  • FIG. 3 is a graph comparing binding constants measured by surface plasma resonance experiments (authentic control: binding constant of IL-4, peptide control: binding constant of AP-1 peptide not bound by protein skeletal parents; Peptide-embeded DIMID: Binding Constant of Protein Skeletal Model Embedded with AP-1 of the Present Invention)
  • FIG. 4 is a photograph showing experimental results of measuring the effect of inhibiting the signal transduction of the IL-4 receptor of the protein skeletal hairs of the present invention in which AP-1 is embedded (IL4: Intertokin 4, API: protein skeletal hairs). Unbound API, AP1-DIMID: API with protein backbone module, STAT6: level of STAT6, pSTAT6: level of phosphorylated STAT6).
  • FIG. 5 is a photograph of cellular imaging experiments measuring the improvement of detection ability of the protein framework module of the present invention in which AP-1 is embedded (DAPI: 4,, 6-diamidino-2-phenyl indole (DAPI) Stained with, FITC: Fluorescein Isothiocyanate labeled protein backbones, MERGE: DAPI and FITC photographs combined, AP1-DIMID-FITC: FITC labeled protein backbones, AP1-FITC: FITC-labeled AP-1, Control DMID-FITC: FTTC-labeled protein backbone model without AP-1 incorporating FITC-labeled FITC-labeled peptide in the NSSSVDK sequence.
  • DAPI 4,, 6-diamidino-2-phenyl indole (DAPI) Stained with, FITC: Fluorescein Isothiocyanate labeled protein backbones, MERGE: DAPI and FITC
  • Figure 6 is a photograph of the results of animal imaging experiments to measure the improvement of detection ability of the protein skeletal hair of the present invention in which AP-1 is embedded (Scafold control: mice injected with protein skeletal hair not embedded in AP-1) , AP-1 Peptide: mice injected with AP-1 peptide, DMID AP-1: mice injected with DMID AP-1). [Form for implementation of invention]
  • the modular domains present in a large amount of protein domains expressed in humans are a) high affinity, b) low.
  • EGFL (EGF-like) domains were selected based on the conditions of molecular weight, c) low imraunogenicity, d) abundance in humans, and e) capable of tertiary conformational verification.
  • EGFL consists of 40 amino acids present in a number of plasma proteins, and has low molecular weight, high safety, and low immunity.
  • Two beta-sheet and three di ⁇ sulfide bonded three-dimensional structure as a simple structure consisting of the verification is possible and to the loop does not affect the structure of the active polypeptide present easy insert, so is suitable as a beam and then DMID.
  • ⁇ i95> BLAST and AlignMaster programs were used for three-dimensional modeling of EGFL domains.
  • the structure of the human EGFL domain was provided by Protein DataBank.
  • nnPr ed i c www. Cmphar m. Uc s f. Edu / ⁇ nom i / nnpr ed i c t. Ht m 1
  • jppred nnPr ed i c
  • the EGFL domain (4621-4869) of the Stabilin 2 protein was cloned into the BamHl and Xhol sites of pET32a (Novagen), an E-col i expression vector. Expression protein was prepared. Mutation of GCTGAC (4639-4644) of EGFL domain to GTCGAC to produce restriction enzyme Sail site and mutation of AAAGCA (4703-4708) to MGCTT to prepare restriction enzyme Hindl ll si te to insert active polypeptide A DMID-Sal-Hindlll cassette was prepared.
  • TP Tryptone phosphate
  • the culture medium was cooled to 20 ° C and induced protein expression for 12 hours with 0.2 mM IPTG (Isopropyl-p -D-thiogalactoside).
  • IPTG Isopropyl-p -D-thiogalactoside
  • the stored cells were resuspended in 10 ml of 2X PBS containing ImM PMSF, 0.5 mM DTT, and then ultrasonically pulverized under the conditions of Duty cycle: 30% and Output: 2.5 for 5 min. The supernatant was taken by centrifuging the pulverized cells at 15,000g for 10 minutes.
  • Ni-NTN column (amersham equilibrated with 2XPBS Buf fer the recovered supernatant)
  • the binding constants of naked API and DMID-API were measured by surface plasmon resonance analysis using Biacore 2000.
  • HBS buffer 10 mM HEPES, H 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20
  • the Biacore CM5 chip surface was coated according to the manufacturer's method (2500 RU).
  • the binding constant was determined using the Biaevaluation 2.0 program.
  • the phosphorylation of Stat protein induced by binding between IL-4 receptors was performed by Western blot method using Rabbit polyclonal antibody anti ⁇ STAT6 (Cell Signaling, # 9362) and rabbit polyclonal antibody anti-pTyr641-STAT6 (Cell Signaling, # 9361). Measured with
  • the DMID-AP1 of the present invention is compared to the API.
  • DMID-AP1 protein-internal protein
  • naked API in H226 cell line and IL-4 of DMID-AP1 The binding ability to the receptor was measured using fluorescently labeled proteins.
  • AP-1 peptide black was dialyzed at 4 ° C with purified DMID-AP1 at 50 ° C with dialysis stratified solution (50 mM Boric acid, 150 mM NaCl, ImM EDTA, pH 9.0) and 37 ° C with fluorescent material Fluorescein Isothiocyanate (FITC) (sigma). Labeling was repeated at C for one hour.
  • FITC Fluorescein Isothiocyanate
  • the reaction was stopped by adding 50 raM Glycine at 37 ° C. for 5 minutes and dialyzed at 4 ° C. with dialysis buffer (20 mM Tris HC1, pH 8.0). The same amount of control peptide, AP-l, and DMID—Apl was added to H226 cells to bind, washed, and fixed with 4% paraformaldehyde (PFA). The nuclei were 4 ', 6-di am idi ⁇ -2-pheny After staining with 1 i ndo 1 e (DAPI), fluorescence microscopy (Zeiss, Oberkochen, Germany) was observed.
  • DAPI 1 i ndo 1 e
  • ⁇ 23i> 1 ⁇ 10 7 H226 cells were suspended in culture medium, and then subcutaneous tumor models were prepared by subcutaneous injection into the right thigh of female BALB / c nude mice 5-6 weeks old. About three weeks later, the tumors were used in the experiment when the tumors became 0.1 to lcm in size.
  • Tumor xenograft mice were anaesthetized and injected with fluorescent conjugated AP-1 peptide and DMID-API 150 ug into the tail vein. Tumor targeting function of the fluorescently labeled peptide was analyzed using Optics.
  • the present invention relates to a novel protein backbone model that enhances the binding affinity or binding specificity of an active polypeptide, more specifically SEQ ID NO: 1.

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PCT/KR2012/002958 2011-04-18 2012-04-18 활성 폴리펩타이드의 결합 친화력 및 결합 특이성을 증강시키는 단백질 골격 모듈 WO2012144799A2 (ko)

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CN201280029758.7A CN103703022A (zh) 2011-04-18 2012-04-18 使活性多肽的结合亲和力和结合特异性增强的蛋白质骨架模块
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